Hindawi BioMed Research International Volume 2021, Article ID 7509825, 6 pages https://doi.org/10.1155/2021/7509825

Research Article Mutational Analysis of Myoclonin1 in Pakistani Juvenile Myoclonic Epilepsy Patients

Tayyaba Saleem , Arooj Mustafa , Nadeem Sheikh , Maryam Mukhtar , Mavra Irfan , and Saira Kainat Suqaina

Cell and Molecular Biology Laboratory, Department of Zoology, University of the Punjab, Lahore 54590, Pakistan

Correspondence should be addressed to Nadeem Sheikh; [email protected]

Received 22 July 2020; Revised 3 February 2021; Accepted 10 April 2021; Published 21 April 2021

Academic Editor: Bilal ig

Copyright © 2021 Tayyaba Saleem et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Juvenile myoclonic epilepsy (JME) is the most prevalent and genetically heterogeneous form of epilepsy and accounts for 10–30% of all the cases worldwide. Ef-hand domain- (c-terminal-) containing 1 (EFHC1) encodes for a nonion channel protein and in this gene have been extensively reported in different populations to play a causative role in JME. Linkage between JME and 6p11-12 locus has already been confirmed in Mexican and Dutch families. A case-control study was conducted on Pakistani JME patients for the first time, aimed at finding out EFHC1 mutations that have been reported in different populations. For this purpose, 66 clinically diagnosed JME patients and 108 control subjects were included in the study. Blood samples were collected from all the participants, and DNA was isolated from the lymphocytes by the modified organic method. Total 3 exons of EFHC1, harboring extensively reported mutations, were selected for genotypic analysis. We identified three heterozygous variants, R159W, V460A, P436P, and one insertion in the current study. V460A, an uncommon variant identified herein, has recently been reported in public databases in an unphenotyped American individual. This missense variant was found in 3 Pakistani JME patients from 2 unrelated families. However, in silico analysis showed that V460A may possibly be a neutral variant. While the absence of a majority of previously reported mutations in our population suggests that most of the mutations of EFHC1 are confined to particular ethnicities and are not evenly distributed across the world. However, to imply the causation, the whole gene and larger number of JME patients should be screened in this understudied population.

1. Introduction regular basis but upon discontinuation of medication, a high recurrence rate has been reported in various studies [5, 6]. Juvenile myoclonic epilepsy (JME) is the most prevalent form JME seizures occur in adolescents with the typical onset of genetic generalized epilepsy and accounts for at least age of 12-18 years [7]. It has been found more prevalent 10–30% of all epilepsies [1, 2]. JME alone is responsible for in females than in males (23). Till date, mutations in eight 2 million cases in India [3]. No epidemiological data of Mendelian (gamma-aminobutyric acid receptor JME is present for Pakistan, so the exact prevalence is not subunit alpha-1; GABRA1, calcium-sensing receptor; CASR, known yet. JME is typified by myoclonic jerks that predom- gamma-aminobutyric acid receptor, delta; GABRD, calcium inantly occur after waking, generalized tonic–clonic (GTC) channel, voltage-dependent, beta-4 subunit; CACNB4, ef- seizures, and infrequent absence seizures [4]. Epileptologists hand domain- (c-terminal-) containing protein 2; EFHC2, follow the varied criteria for JME diagnosis but the key diag- bromodomain-containing protein 2; BRD2, sodium chan- nostic feature, according to all epileptologists is the same that nel, voltage-gated, type i, beta subunit; SCN1B, and ef- all JME patients experience myoclonic seizures with or with- hand domain (c-terminal)-containing protein 1; EFHC1) out GTCS and absence seizure in the early morning. Typical have been identified which are linked with JME [8, 9]. So EEG symptom for JME is considered as polyspike wave pat- far, heterozygous mutations in the coding sequence of tern of >4 Hz with normal background activity. Seizures are EFHC1/Myoclonin1 are persistent, causing JME including well controlled when the patient follows the prescription on autosomal dominant, singleton, and sporadic cases in 2 BioMed Research International various independent families around the world [7, 10, 11]. Table 1: Clinical characters of the JME patients. Genetic studies have affirmed that mutations in EFHC1 con- N stitute 3 to 9% of all the JME cases all over the world [12]. Character EFHC1 gene consisting of 11 exons encodes a 70 kDa protein Gender 66 of 640 amino acids containing three DM10 domains, a motif Female 27 with unknown function, and an EF-hand Ca2+-binding Male 39 motif. EFHC1 modulates the apoptotic activity by interacting mean ± SD 17 ± 3:33 with R-type voltage-dependent calcium channels. Thus, the Age years ( ) mutations in EFHC1 interfere with the apoptotic activity of Female 16 ± 3:12 this gene by increasing the neuronal density leading to the Male 15 ± 3:24 production of hyperexcitable circuits [13]. Interestingly, EFHC1 mutations are not limited to JME Seizure type but it has also been identified in different idiopathic general- Myoclonic seizures 66 ized epilepsies and temporal lobe epilepsy. EFHC1 mutations GTCS 50 may be considered pleiotropic due to their involvement in Absence seizures 6 various epilepsy phenotypes [14]. EFHC1 (a microtubule- Absence+myoclonic 2 associated protein) plays a vital role in radial migration and Absence + myoclonic + GTCS 4 cell division during cerebral corticogenesis. Functional anal- ysis of various mutations has established that mutant EFHC1 Myoclonic+GTCS 46 impairs mitotic spindle organization and affects the mor- Myoclonic seizures alone 14 phology of radial glia that further leads to disruption of the Distribution of myoclonic jerks radial and tangential migration. Mutations in the EFHC1 Upper extremities 57 gene potentially produce structural brain anomalies due to Lower extremities 09 disruption in brain development [15]. Several studies have indicated that mutations in neuro- Precipitating factors transmitter receptors and ion channel genes are linked with Sleep deprivation 39 JME. The mutations that have been reported in these genes Fatigue 21 were private mutations, only found in single families and Stress 11 typically of de novo origin, that are not spotted in biological parents. Importantly, these de novo mutations (DNMs) were Family history not detected in other family-based association studies for 1st-degree relatives 11 JME in the same or different ethnicities [16]. Consequently, 2nd-degree relatives 17 mutations in genes coding for ion channel cannot be accounted as the common cause of JME. EFHC1 is consid- ered as a potential candidate gene for JME and has been is indispensable to ascribe causation [20]. Therefore, to found very interesting in many aspects. First, EFHC1 is the ascertain whether the different variants of the EFHC1 gene only gene in which mutations were found in many unrelated contribute to JME in Pakistani patients, we genotyped 3 families with JME across the world, and second, no ion chan- important exons of this gene on which mutations had been nel protein is coded by it as usually the case in epilepsy [17]. reported extensively in previous genetic studies. This presents a new perspective to the pathophysiology of JME and genetic epilepsy as a whole. However, Pinto et al. 2. Materials and Methods found 6p12–11 loci very heterogeneous and reported EFHC1 mutations nonexistent in 112 Dutch patients of JME [18]. 2.1. Subjects. 66 Pakistani patients with JME were selected for Hence, it can be presumed that JME may be caused by muta- the current study from two different hospitals of Punjab, tions in multiple genes, which may vary between populations Pakistan, who were visiting the outpatient department from of different ethnic origins. Mapping of 6p12–11 loci in a large September 2018 to November 2019. All JME patients were Belize family with JME revealed a common EFHC1 polymor- diagnosed by neurologists by the following criteria. The onset phism present in higher frequency than in healthy individ- of myoclonic seizures varied between 8 and 20 years age uals, cosegregating with juvenile myoclonic epilepsy. When bracket. Patients experienced short bilateral myoclonic jerks the cell death analysis mediated by EFHC1 was judged, it of shoulders and arms without losing consciousness just after was evident that this common polymorphism had no effect awakening. Interictal electroencephalography (EEG) of the on protein function, prospecting the involvement of other patients showed diffused, bilateral synchronous, and 4-6 Hz nearby mutations accountable for JME in this family [19]. polyspike waves with normal background, provoked by Several studies had questioned the -specific patho- photic stimulation [21, 22]. Furthermore, patients with focal genic effects of the EFHC1 gene linked to JME. Subaran seizures, mental retardation, or with a suggestion of any et al. highlighted that the pathogenicity of EFHC1 mutations degenerative disease were excluded from this study. This may depend on heterogeneity in terms of the genetic back- study was approved by the Bioethics Committee of Univer- ground of the ethnic group considered for the study. Fur- sity of the Punjab, Lahore, Pakistan. Written informed con- thermore, this study cautions us that compelling evidence sent was signed by all the patients/guardians before taking BioMed Research International 3

Table 2: Variants identified by sequencing the exons of EFHC1 in Pakistani JME patients.

Genotype counts/(no. of No. Region NCBI dbSNP ID Nucleotide change Amino acid change Molecular consequence subjects) JME probands Control subjects 1 Exon 3 rs3804506 475C > T R159W Missense 19/66 11/108 2 Intron 5 rs1581829739 c:723 + 18 723 + 19insG N/A Unknown 1/66 0/108 3 Exon 8 rs764251038 1436 T > C V460A Missense 3/66 0/108 4 Exon 8 rs1581846971 1365T > C P436P Synonymous 2/66 0/108 the blood sample. Moreover, 108 control subjects of the same tively. No other JME patients or control subjects carried these ethnicity and geographic origin were included in the study. variants. However, we were not able to collect the blood All the control subjects were healthy and had no history of samples of the whole family of these probands. Sequences any neurological disorder. of both of these novel variants were submitted to ClinVar- NCBI and assigned with accession number. Reference SNP 2.2. EFHC1 Genotyping. Peripheral venous blood was col- (rs or RefSNP) number assigned by dbSNP to these variants lected from all the participants of this study in EDTA- is also given in Table 2. Electropherograms of all the variants coated tubes. DNA extraction was done by the modified detected in the current study are given in Figure 1 with wild organic method [12]. Each DNA sample was subjected to type and mutant alleles. quantification and qualification before amplifying the PCR products. Total 3 exons carrying common SNPs rs3804505, 4. Discussion rs3804506 (exon 3), rs137852777 (exon 4), and rs1266787 (exon 8) of EFHC1 were targeted for mutational analysis in Our study showed slight male predominance in JME Pakistani JME patients. To amplify the PCR products, previ- patients. Gender distribution is cogitated to be equal but ously reported primers were used [23]. The purification of some studies have manifested female preponderance [24]. amplicons was done by using the purification kit of GeneJET Total 674 single nucleotide polymorphisms including 132 PCR (Thermo Scientific™ #K0702) and sequenced commer- exonic variants have been identified in the myoclonin1 gene cially. All the sequence electropherograms of cases and con- and can be accessed through the database trols were analyzed by BioEdit 7.2 software to find out (GENOME 1000). Till date, many pathogenic mutations, variations. NCBI-BLAST was also performed to locate any including missense variant, deletions in the promoter region, change in the sequenced DNA samples. MEGA6 software nonsense variant, and frameshift variant, have been studied was used to find the consequent amino acid changes. SNPs- in different populations around the globe but not a single GO and PhD-SNP were used for the in silico analysis of the study from Pakistan added data to this database [10, 22]. This identified variants. is the first study that investigated the variants of the EFHC1 gene with reference to JME in the Pakistani population. 3. Results JME prevalence has not yet been evaluated in Pakistan, but on the base of our experience of blood sampling for JME 3.1. Clinical Characteristics. Our cohort of 66 JME patients patients, we can presume that JME has a lower prevalence contained 39 (59%) males and 27 (40%) females. The average compared to other idiopathic epilepsies in this Asian region. age of onset for JME was observed 12 years varied between 10 We evaluated 66 Pakistani JME patients for the EFHC1 vari- and 19 years. Whereas in the control group, there were 64 ants and unearthed 4 variants in total 3 exons and their males and 44 females. The mean age of the control group immediately adjacent intronic regions. The identified vari- was found 15 years. Clinical characteristics of the JME ants are presented in Figure 2 along with previously reported patients are given in Table 1. mutations. All these variants were considered as singleton as the mode of inheritance was not determined due to lack of 3.2. Mutation Analysis. We identified 4 different variants in blood samples from families. The result of this study are neg- 66 JME patients, two of which were benign variants that ative for R182H (exon 3), D210N, R221C, R221H, F229L had not been reported in dbSNP or any other database and (exon 4), and M448T (exon 8) that have been reportedly are listed in Table 1. Two heterozygous variants were found in Hispanic, Indian, Dutch, African American, and detected in exon 8; one in exon 3, while in noncoding region Caucasian JME patients [13, 18, 23]. It can be conferred that adjacent to exon 4 of EFHC1, 1 bp insertion was found. Mis- EFHC1 mutations are not evenly distributed in different sense heterozygous variant c:475C > T (rs3804506) found in ethnicities. exon 3 led to the change of arginine into tryptophan at 159 Missense variant R159W located on exon 3 was found positions in the protein. It was observed in heterozygous both in JME patients and healthy controls, so it was consid- form in JME patients and healthy control subjects. While a ered as a polymorphism. Consistent with the findings of very rare heterozygous missense variant c:1436 T > C Ma et al., the frequency of this polymorphism did not differ (rs764251038) and a benign synonymous variant c:1365 T in both subjects in the Pakistani population, so it cannot be >Cwere detected in 3 and 2 Pakistani JME patients, respec- considered as a major predisposing factor for JME [25]. In 4 BioMed Research International

190 190 T G A CCGGG AATTG C GGG

(a) (b) 190 190 G A AAACC C G A AACCG C

(c) (d) 100 TTTCC G TTTTTCC G

(e) (f)

C A G T G G C A G T G G

(g) (h)

Figure 1: Electropherograms of identified variants. (a) Wild type and (b) R159W; (c) wild type and (d) c:723 + 18 723 + 19insG; (e) wild type and (f) P436P; (g) wild type and (h) V460A. another study conducted on the Hispanic population, it was in one JME patient of this study. This insertion was not observed that R159W occurred almost at equal frequency reported before the current study in any ethnicity and possi- in patients and control subjects [23]. Pinto et al. also bly is benign in nature. A very rare heterozygous missense observed this polymorphism in Dutch families but failed to variant V460A was found in 3 JME patients belonging to find any statistically significant difference at allelic and geno- two unrelated families. Two of these JME patients were sib- typic level [18]. In the current study, 1 bp insertion was also lings. This family had 3 JME affected individuals with unaf- found in the intronic region immediately adjacent to exon 4 fected parents, but the unavailability of blood samples from BioMed Research International 5

DM10 DM10 DM10 EFh COOH NH2

1 93-198 239-359 416-520 578-606 640

(a) R260Q F229L R118C C259Y L9L R221H R294H E357K V460A P436P P77T R159W I619L R153Q

1 2 3 4 5 6 7 8 9 10 11

T30T R221C R244STOP D210N T252K N606N 2014T>C M448T I174V R182H/L D253Y A394S c.723+18_723+19insG (b)

Figure 2: (a) Diagrammatic representation of EFHC1 protein. (b) Genomic arrangement of myoclonin1 gene. Previously reported mutations along with novel variants identified in Pakistani JME patients are shown in different exons. Novel variants detected in the Pakistani patients are given in bold type. all the members limited our effort to study the inheritance Conflicts of Interest pattern of this very rare variant. V460A is a coding missense fl variant recently been submitted to The Single Nucleotide The authors declare that there is no con ict of interest Polymorphism Database (dbSNP) by The Exome Aggrega- regarding the publication of this paper. tion Consortium (ExAC) and The Genome Aggregation Database (gnomAD) Exomes in unphenotyped individuals. Acknowledgments It was found neutral variant when pathogenicity of this vari- ant was checked by SNPs-GO, PhD-SNP, and several other The authors are thankful to all the participants of this study. in silico tools. One novel synonymous variant (P436P) was This work was supported by Higher Education Commission, also identified in the two JME patients of this study and Pakistan (No.: 8369/Punjab/NRPU/R&D/HEC/2017) under was not found in any control subject. National Research Program for Universities. To imply the causality, the altered function of protein caused by any of the novel variant should be monitored by References biological assays. The same notion can be expanded to all the novel missense variants identified in any earlier studies, [1] A. Delgado-Escueta and F. Enrile-Bacsal, “Juvenile myoclonic which are considered pathogenic on the basis of cosegrega- epilepsy of Janz,” Neurology, vol. 34, no. 3, pp. 285–294, 1984. tion and prevalence among JME patients. [2] A. V. Delgado-Escueta, “Advances in genetics of juvenile myo- clonic epilepsies,” Epilepsy Currents, vol. 7, no. 3, pp. 61–67, 5. Conclusion 2007. [3] K. Haug, M. Warnstedt, A. K. Alekov et al., Retraction: muta- Uneven distribution of EFHC1 mutations among different tions in CLCN2 encoding a voltage-gated chloride channel are ethnicities explains the importance of the genetic back- associated with idiopathic generalized epilepsies, Nature Pub- ground of the patient for a mutation to play a causative role. lishing Group, 2009. “ We identified only one previously reported pathogenic vari- [4] D. G. Trenité, B. Schmitz, D. Janz et al., Consensus on diagno- ant in the current study, indicating that some other genes at sis and management of JME: from founder's observations to current trends,” Epilepsy & Behavior, vol. 28, pp. S87–S90, the EJM1 locus might be involved in JME susceptibility in 2013. this population. In the present study, only 3 exons of the [5] M. Pavlović, N. Jović, and T. Pekmezović, “Antiepileptic drugs EFHC1 gene were analyzed, other exons and the promoter withdrawal in patients with idiopathic generalized epilepsy,” region of this gene remained to be evaluated, which may har- Seizure, vol. 20, no. 7, pp. 520–525, 2011. bor the disease-causing mutations. Assertion of EFHC1 [6] B. P. Santos, C. R. Marinho, T. E. Marques et al., “Genetic sus- genetic contribution to JME necessitates the evaluation of ceptibility in juvenile myoclonic epilepsy: systematic review of complete gene at a larger scale coinciding with the inheri- genetic association studies,” PloS one, vol. 12, no. 6, article tance pattern in Pakistani patients. e0179629, 2017. [7] I. Berger, T. Dor, J. Halvardson et al., “Intractable epilepsy of Data Availability infancy due to homozygous mutation in the EFHC1 gene,” Epilepsia, vol. 53, no. 8, pp. 1436–1440, 2012. The data used to support the findings of this study is available [8] M. Avoli and H. Herbert, “Jasper and the basic mechanisms of upon request from the corresponding author. the epilepsies,” in Jasper's basic mechanisms of the epilepsies, 6 BioMed Research International

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